CREB is a transcription factor that binds to CRE. The Ser 133 of CREB is phosphorylated and then activated by cyclic AMP-dependent protein kinase. CRE is located in the transcription regulatory sites of gene clusters whose expression is enhanced by cAMP (somatostatin, c-fos, etc.), and is an indispensable component for the cAMP-induced induction.
CREB whose serine has been phosphorylated binds to transcription regulatory factors, mainly CREB binding proteins (CBPs), and has increased binding activity (Shaywitz, A. J. et al., Annu. Rev. Biochem. 68:821 (1999)). Furthermore, an increase of CREB's transcriptional activity has been observed on depolarization due to high K+ concentration (Beitmer-Johnson, D. et al., J. Biol. Chem. 273:19834-19839 (1998)), or synapse stimulation caused by long-term enhancement (LTP) (Deisseroth, K. et al., Neuron 16:89-101 (1996)). Moreover, glutamic acid-induced neuronal calcium signals are known to increase the CREB's transcriptional activity evoked by synaptic activity (Hardingham, G. E. et al., Nat. Neurosci. 4:261-267 (2001)). In addition, results of behavioral memory experiments on Sea Hares (Aplysia) and rats revealed that CREB phosphorylation is important for memory formation (Silva, A. J. et al., Annu. Rev. Neurosci. 21:127 (1998)). Furthermore, in Alzheimer's dementia, the amount of phosphorylated CREB in the brain is significantly decreased (Yamamoto-Sasaki, M. et al., Brain Res. 824:300-303 (1999)). These findings suggest that increased CREB activity is related to memory formation.
Recently, increased CREB activity was shown to be linked to the anti-apoptosis activity of neurons. In pathological models of cerebral infarction and such, CREB phosphorylated regions were observed in the penumbra region near the infarct (Tanaka, K. et al., Brain Res. 818: 520-526 (1999) ). The term “penumbra region” refers to a region around the cerebral infarcted lesion, which has decreased blood flow but continuous neural activity. In general, when conditions of insufficient blood flow to the penumbra region continue for 48 hours or more, neuron death can be observed. This fact has also been observed in experimental systems on a cellular level. An increase in CREB's transcriptional activity due to hypoxia (Beitmer-Johnson, D. et al., supra) andlowglucose stimulation (Ricco, A. et al., Science 286:2358-2361 (1999) ) in cultured neurons has also been confirmed. Furthermore, a survival effect due to increased CREB transcription has been confirmed in the case of neuron death caused by the removal of nutritional factors (Ricco, A. et al., supra).
CBP functions as a co-activator of CREB. CBP is known to bind to both phosphorylated CREB and to general transcription factor TFII B, to promote CREB's transcriptional activity (Shaywitz, A. J. et al ., supra). Huntingtin, the product of the IT15 gene which causes Huntington's disease, and atrophin-1, the product of the gene which causes dentatorubropallidoluysian atrophy, actively bind to CBP and suppress its function (Nucifora Jr., F. C. et al., Science 291: 2423-2428 (2001) ). Specifically, huntingtin and atrophin-1 have been known to suppress CREB's transcriptional activity via CBP, and to damage neurons by preventing the transcription of target genes known to be decisive in neuron survival.
In general, various cellular phenomena are known to be regulated through phosphorylation (or dephosphorylation) by protein kinases and phosphatases. Already revealed examples of such phenomena are, mechanisms regulated by phosphorylation, such as contraction, membrane transport, glycogen metabolism, and cell division. Since CBP binds to phosphorylated CREB to promote transcriptional activity, phosphatase-related genes are also thought to be involved in CREB's transcriptional activity. It is highly probable that phosphatase-related genes act on the dephosphorylation of CREB to suppress its transcriptional activity. Therefore, increased CREB's transcriptional activity is suggested to have an ameliorative effect on memory and nerve degeneration.
Protein phosphatase-1 (PP-1), a typical Ser/Thr phosphatase, is known as a major down regulator of CREB activity due to cAMP stimulation (Hagiwara, M. et al., Cell 70:105-113 (1992)). PP-1 activity was reported to be inhibited by phosphorylated Inhibitor-1 (I-1) (Cohen, P., Annu. Rev. Biochem. 58:453-508 (1989)). This report suggested that cellular cAMP response may be amplified by blocking CREB dephosphorylation. However, other than PP-1, phosphatases that down-regulate CREB activity due to cAMP stimulation are not presently known.